Prodigiosin as well as production strain, preparation method and application thereof

Abstract

The invention belongs to the technical field of biology, and discloses prodigiosin as well as a production strain, a preparation method and application thereof. The production strain of the prodigiosin provided by the invention is serratia marcescens MMOG2448, and the preservation number of the serratia marcescens MMOG2448 is GDMCC 65757. The production strain has excellent prodigiosin synthesis capability and prodigiosin cell accumulation capability, the yield of the prodigiosin is as high as 1458.858 mg / L, the requirement of industrial production of the prodigiosin can be well met, and the prodigiosin has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to prodigiosin and a production strain, a preparation method and an application thereof. Background Art

[0002] Prodigiosin is a linear cyclic compound with a tripyrrole ring. It is a red pigment produced by the secondary metabolism of microorganisms and exists in the cell wall of microbial cells. Prodigiosin has multiple biological activities such as anticancer, antibacterial and immunosuppressive, and has received more and more attention.

[0003] At present, prodigiosin is mainly synthesized by microbial method, chemical synthesis method and other methods. Among them, the microbial method has the advantages of low cost, mild conditions and no secondary pollution, and is the main way to synthesize prodigiosin. Specifically, microorganisms of Vibrio, Streptomyces and Serratia are used to ferment to obtain prodigiosin. However, the prodigiosin yield of the microorganisms used in the existing microbial method is low, which cannot well meet the requirements of industrial production of prodigiosin and has certain limitations. Summary of the invention

[0004] The first object of the present invention is to provide a prodigiosin production strain, which is Serratia marcescens MMOG2448 with a deposit number of GDMCC 65757. The production strain has excellent prodigiosin synthesis ability and prodigiosin cell accumulation ability, and the prodigiosin yield is as high as 1458.858 mg / L, which can well meet the requirements of industrial production of prodigiosin and has good application prospects.

[0005] The second object of the present invention is to provide a method for preparing prodigiosin, which specifically comprises: S1, taking the above-mentioned prodigiosin production strain for fermentation culture to obtain a fermentation culture liquid; S2, taking the fermentation culture liquid for purification treatment to obtain the prodigiosin.

[0006] In the present invention, in step S1, the culture medium for fermentation culture is preferably selected from one or more of G2 culture medium, PG culture medium, NB culture medium, NBM culture medium, PBO culture medium, SP culture medium, GPK culture medium, and Brown culture medium. At this time, the production strain has excellent prodigiosin synthesis and accumulation capabilities in the fermentation culture medium. Among them, the G2 culture medium specifically includes 5-20g / L glycerol, 1-5g / L peptone and 1-5g / L yeast powder. The PG culture medium specifically includes 1-10g / L glycerol and 1-10g / L peptone. The NB culture medium specifically includes 5-20g / L peptone, 1-10g / L sodium chloride and 1-5g / L yeast powder. The NBM culture medium specifically includes 10-20g / L peptone, 1-10g / L sodium chloride, 1-5g / L yeast powder and 1-10g / L maltose. The PBO medium specifically includes 10-30 g / L peanut powder, 10-20 g / L yeast powder and 1-2% (v / v) olive oil. The SP medium specifically includes 15-20 g / L starch, 10-15 g / L peptone, 0.1-1 g / L manganese sulfate, 0.1-1 g / L magnesium sulfate, 5-10 g / L calcium chloride and 0.1-0.3 g / L ferrous sulfate. The GPK medium specifically includes 1-10 g / L glycine, 1-10 g / L sucrose, 1-10 g / L peptone, and 1-5 g / L potassium dihydrogen phosphate. The Brown medium includes 0.1-0.5 g / L proline, 5-20 g / L peptone, 1-10 g / L sodium chloride, 1-5 g / L yeast powder and 10-30 g / L brown sugar.

[0007] In some specific embodiments, in step S1, the inoculation amount of the prodigiosin producing strain is preferably 10-10 CFU / mL, such as 10 5 CFU / mL, 1.5×10 5 CFU / mL, 9×10 5 CFU / mL, 10 6 CFU / mL, 5×10 6 CFU / mL, 10 7 CFU / mL or any value therebetween. At this time, the production strain in the fermentation culture system has a relatively ideal physiological state, and can well achieve high-yield synthesis of prodigiosin and cell accumulation.

[0008] In some specific embodiments, in step S1, the fermentation culture temperature is preferably 25-30°C, such as 25°C, 26.5°C, 27°C, 28°C, 30°C or any value therebetween; the fermentation time is preferably 24-72h, such as 24h, 30h, 36h, 48h, 60h, 72h or any value therebetween.

[0009] In the present invention, in step S2, the purification treatment includes: (1) taking the fermentation culture liquid and performing freeze-drying treatment to obtain freeze-dried powder; (2) taking the freeze-dried powder and mixing it with acidic formaldehyde, performing ultrasonic crushing, light-proofing and standing treatment and rotary evaporation treatment I to obtain the substance to be extracted; (3) taking the substance to be extracted and mixing it with water to obtain the liquid to be extracted, taking the liquid to be extracted and performing extraction treatment and rotary evaporation treatment II with an extractant to obtain an extracted dry substance; (4) taking the extracted dry substance and mixing it with acidic formaldehyde, performing filtering treatment and rotary evaporation treatment III to obtain the prodigiosin. At this time, the purification treatment can effectively remove other impurities in the fermentation culture liquid, thereby obtaining high-purity prodigiosin.

[0010] In the present invention, in the purification process, in step (1), the freeze-drying process is a technical means conventionally used in the art, which is limited to achieving drying and powdering of substances, and the present invention does not impose any particular limitation thereto.

[0011] In the present invention, the freeze-drying process may specifically include: taking the fermentation culture solution and performing a first freezing process, a second freezing process and vacuum drying to obtain the freeze-dried powder.

[0012] In some specific embodiments, in the freeze-drying treatment, the temperature of the first freezing treatment is preferably -25 to -18°C, such as -25°C, -23°C, -20°C, -19°C, -18°C or any value therebetween; the time is preferably 12 to 48h, such as 12h, 13h, 14h, 18h, 24h, 36h, 48h or any value therebetween.

[0013] In some specific embodiments, in the freeze-drying treatment, the temperature of the second freezing treatment is preferably -85 to -75°C, such as -85°C, -83°C, -80°C, -79°C, -75°C or any value therebetween; the time is preferably 12 to 48h, such as 12h, 13h, 14h, 18h, 24h, 36h, 48h or any value therebetween.

[0014] In some specific embodiments, in the freeze-drying process, the vacuum drying temperature is preferably -85 to -75°C, such as -85°C, -83°C, -80°C, -79°C, -75°C or any value therebetween; the air pressure is preferably 0.1 to 0.2 Pa, such as 0.1 Pa, 0.13 Pa, 0.15 Pa, 0.18 Pa, 0.2 Pa or any value therebetween; the time is preferably 48 to 72 h, such as 48 h, 50 h, 60 h, 68.5 h, 72 h or any value therebetween;

[0015] In some specific embodiments, in the purification process, in step (2), the pH value of the acidic formaldehyde is preferably 2 to 3.5, such as 2, 2.3, 2.7, 2.9, 3, 3.2, .35 or any value therebetween.

[0016] In some specific embodiments, in the purification process, in step (2), the added mass volume ratio of the lyophilized powder to the acidic formaldehyde is preferably 1 g:(5-20) mL, such as 1 g:5 mL, 1 g:8 mL, 1 g:10 mL, 1 g:20 mL or any value therebetween.

[0017] In some specific embodiments, in the purification treatment, in step (2), the temperature of the light-proof static treatment is preferably 0-5°C, such as 0°C, 1°C, 3°C, 4°C, 5°C or any value therebetween; the time is preferably 12-48h, such as 12h, 18h, 24h, 36h, 48h or any value therebetween.

[0018] In some specific embodiments, in the purification treatment, in step (2), the temperature of the rotary evaporation treatment I is preferably 30-40°C, such as 30°C, 31.5°C, 33°C, 35°C, 36.5°C, 38°C, 40°C or any value therebetween; the air pressure is preferably 100-200 mbar, such as 100 mbar, 115 mbar, 125 mbar, 130 mbar, 148 mbar, 156 mbar, 170 mbar, 180 mbar, 190 mbar, 200 mbar or any value therebetween; and the time is preferably 30-60 min, such as 30 min, 35 min, 45 min, 50 min, 55 min, 60 min or any value therebetween.

[0019] In some specific embodiments, in the purification treatment, in step (3), the added mass volume ratio of the substance to be extracted and water is 1g:(10-50)mL, such as 1g:10mL, 1g:20mL, 1g:38mL, 1g:50mL or any value therebetween; and the added volume ratio of the liquid to be extracted and the extractant is preferably 1:(0.8-1.2), such as 1:0.8, 1:0.9, 1:1, 1:1.2 or any value therebetween.

[0020] In some specific embodiments, in the purification treatment, in step (3), the temperature of the rotary evaporation treatment II is preferably 30-40°C, such as 30°C, 31.5°C, 32°C, 34°C, 38°C, 40°C or any value therebetween; the air pressure is preferably 100-200 mbar, such as 100 mbar, 135 mbar, 148 mbar, 150 mbar, 175 mbar, 190 mbar, 200 mbar or any value therebetween; and the time is preferably 60-120 min, such as 60 min, 80 min, 100 min, 120 min or any value therebetween.

[0021] In some specific embodiments, in the purification treatment, in step (4), the pH value of the acidic formaldehyde is preferably 2.0 to 3.5, such as 2.0, 2.3, 2.5, 2.8, 3, 3.1, 3.4, 3.5 or any value therebetween.

[0022] In some specific embodiments, in the purification treatment, in step (4), the added mass volume ratio of the extracted dry matter to the acidic formaldehyde is preferably 1g:(5-20)mL, such as 1g:5mL, 1g:7.5mL, 1g:15mL, 1g:20mL or any value therebetween.

[0023] In some specific embodiments, in the purification treatment, in step (4), the temperature of the rotary evaporation treatment III is preferably 30-40°C, such as 30°C, 32°C, 34°C, 38°C, 40°C or any value therebetween; the air pressure is preferably 100-200 mbar, such as 100 mbar, 125 mbar, 150 mbar, 175 mbar, 200 mbar or any value therebetween; and the time is preferably 60-90 min, such as 60 min, 65 min, 70 min, 80 min, 90 min or any value therebetween.

[0024] It should be noted that the "I", "II" and "III" in the above-mentioned rotary evaporation treatment I, rotary evaporation treatment II and rotary evaporation treatment III are only for the convenience of description and have no other limiting effect; the conditions adopted for the rotary evaporation treatment I, rotary evaporation treatment II and rotary evaporation treatment III may be completely the same, partially the same or completely different, as long as the separation of the corresponding substances can be achieved, and the present invention does not impose any special limitation on them.

[0025] The third object of the present invention is to provide prodigiosin, which is prepared by the above-mentioned preparation method.

[0026] In the present invention, the structure of prodigiosin is shown in formula (1):

[0027]

[0028] The fourth object of the present invention is to provide the use of the above-mentioned prodigiosin in ferroptosis inhibitors.

[0029] In the present invention, the prodigiosin has a good chelating effect on free or intracellular iron ions, can effectively reduce the iron ion content in cells, and the prodigiosin can affect the expression of intracellular ferroptosis-related markers such as ROS, MDA and GSH, thereby ultimately achieving an excellent ferroptosis inhibition effect, and has great potential for application as a ferroptosis inhibitor.

[0030] The fifth object of the present invention is to provide the use of the above-mentioned prodigiosin in liver protection drugs and / or health products.

[0031] In the present invention, the prodigiosin can alleviate multiple abnormal indicators such as ALT concentration, AST concentration, LDH concentration and HA concentration in the blood samples of mice with non-alcoholic fatty liver disease, thereby achieving the effect of alleviating non-alcoholic fatty liver disease, and has good potential for application as a liver protection drug and / or health product.

[0032] Biological Deposit

[0033] The production strain Serratia marcescens MMOG2448 provided by the present invention has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on January 13, 2025, with a deposit number of GDMCC NO.65757, and the address of the depository is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the depository code is GDMCC-Guangdong Provincial Microbiological Culture Collection Center. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a graph showing the experimental results of the chromatographic column separation of prodigiosin provided in Example 2 of the present invention;

[0035] Figure 2 for Figure 1 Figure 1 is the experimental result of liquid chromatography analysis of the eluate of band A;

[0036] Figure 3 for Figure 1 Figure 1 is the experimental result of liquid chromatography analysis of the eluate of band B;

[0037] Figure 4 for Figure 1 Figure 1 is the experimental result of liquid chromatography analysis of the eluate of band C;

[0038] Figure 5 for Figure 1 Figure 1 is the experimental result of mass spectrometry analysis of the eluate of band B;

[0039] Figure 6 This is a graph showing the experimental results of testing the chelating ability of prodigiosin for iron ions in a solution provided in Example 3 of the present invention;

[0040] Figure 7 FIG2 is the experimental result of testing the chelating ability of prodigiosin for iron ions in solution provided in Example 3 of the present invention;

[0041] Figure 8 This is a graph showing the experimental results of testing the chelating ability of prodigiosin for iron ions in HEK293T cells provided in Example 3 of the present invention;

[0042] Fig. 9 This is a graph showing the experimental results of the test of the inhibitory effect of prodigiosin on ferroptosis of normal human liver cells L02 (treatment time) provided in Example 4 of the present invention (culture time is 6 hours);

[0043] Fig.10 Figure 2 is the experimental result of the test (treatment time) of the inhibitory effect of prodigiosin on ferroptosis of normal human liver cells L02 provided in Example 4 of the present invention (culture time is 12h);

[0044] Fig.11 This is a graph showing the experimental results of the test of the inhibitory effect of prodigiosin on ferroptosis of normal human liver cells L02 (treatment concentration) provided in Example 4 of the present invention;

[0045] Fig.12 This is a graph showing the experimental results of testing the effect of prodigiosin provided in Example 5 of the present invention on the inhibitory effect of hepatocytes on the expression of ferroptosis markers on the iron ion content in hepatocytes;

[0046] Fig.13This is a graph showing the experimental results of testing the effect of prodigiosin provided in Example 5 of the present invention on the inhibitory effect of hepatocytes on the expression of ferroptosis markers on the content of ROS in hepatocytes;

[0047] Fig.14 This is a graph showing the experimental results of testing the effect of prodigiosin provided in Example 5 of the present invention on the inhibitory effect of hepatocytes on the expression of ferroptosis markers on the MDA content in hepatocytes;

[0048] Fig.15 This is a graph showing the experimental results of testing the effect of prodigiosin provided in Example 5 of the present invention on the inhibitory effect of hepatocytes on the expression of ferroptosis markers on the GSH content in hepatocytes;

[0049] Fig.16 This is a graph showing the experimental results of the test of the alleviating effect of prodigiosin on non-alcoholic fatty liver disease provided in Example 6 of the present invention (ALT);

[0050] Fig.17 Figure 2 (AST) is the experimental result of testing the alleviating effect of prodigiosin on non-alcoholic fatty liver disease provided in Example 6 of the present invention;

[0051] Fig.18 Figure 3 (TC) is the experimental result of testing the alleviating effect of prodigiosin on non-alcoholic fatty liver disease provided in Example 6 of the present invention;

[0052] Fig.19 Figure 4 (LDH) is the experimental result of testing the alleviating effect of prodigiosin on non-alcoholic fatty liver disease provided in Example 6 of the present invention;

[0053] Fig. 20 Figure 5 (HDL) is the experimental result of the test of the alleviating effect of prodigiosin on non-alcoholic fatty liver disease provided in Example 6 of the present invention;

[0054] Fig.21 Figure 6 (LDL) is the experimental result of the test of the alleviating effect of prodigiosin on non-alcoholic fatty liver disease provided in Example 6 of the present invention;

[0055] Fig. 22 Figure 7 (Glu) is the experimental result of testing the alleviating effect of prodigiosin on non-alcoholic fatty liver disease provided in Example 6 of the present invention;

[0056] Fig.23 Figure 8 (HA) is the experimental result of testing the alleviating effect of prodigiosin on non-alcoholic fatty hepatitis provided in Example 6 of the present invention;

[0057] Fig.24Figure 9 is the experimental result of testing the alleviating effect of prodigiosin on non-alcoholic fatty liver disease provided in Example 6 of the present invention (PIICP). DETAILED DESCRIPTION

[0058] Embodiments of the present invention are described in detail below, and the examples of the embodiments are intended to be used to explain the present invention, but should not be construed as limiting the present invention. In the embodiments, if specific techniques or conditions are not specified, the techniques or conditions described in the literature in this area or the product specifications are used. If the manufacturer is not specified for reagents or instruments used, they are all conventional products that can be obtained commercially.

[0059] Example 1

[0060] This example is used to illustrate the identification and biological preservation of a strain MMOG2448 obtained from a soil sample in Huli District, Xiamen City, and specifically includes:

[0061] 1. Morphological identification: The MMOG2448 strain was inoculated on a BHIS agar plate for streak activation, and cultured in an incubator at 37°C and 200 rpm until a single colony grew. The plate was subcultured three times, and Gram staining and morphological identification were performed.

[0062] The identification results were as follows: the MMOG2448 strain was a Gram-negative bacterium, the cell morphology was short rod-shaped, the colony diameter was 1 to 3 mm, and the colony color was red.

[0063] 2. Molecular biological identification: (1) Dip a single colony on the BHIS agar plate and inoculate it into BHIS liquid culture medium. Shake evenly and culture it in an incubator at 37°C and 200 rpm for 12 h. Use a bacterial genomic DNA rapid extraction kit and refer to the instructions for extraction to obtain the genomic DNA of the MMOG2448 strain.

[0064] (2) PCR amplification of genomic DNA was performed using 16S rDNA universal primers to obtain PCR products, which were verified by agarose gel electrophoresis and then sent for sequencing. The 16S rDNA universal primer pair and the 16S rDNA sequencing results of the MMOG2448 strain are shown in Table 1.

[0065] Table 1.

[0066]

[0067] (3) The 16S rDNA sequencing results were compared by Blast and a phylogenetic tree was constructed using MEGA6.0. The homology between the MMOG2448 strain and Serratia marcescens was 100%, which was highly similar and was named Serratia marcescens MMOG2448.

[0068] 3. Preservation of production strains

[0069] The MMOG2448 strain was sent to the Guangdong Provincial Microbiological Culture Collection Center for preservation and confirmed to be alive on January 13, 2025, with the collection number GDMCC 65757.

[0070] Example 2

[0071] This embodiment is used to illustrate a preparation method of prodigiosin, which comprises the following steps:

[0072] S1. Fermentation culture of production strains: according to 10 6 Inoculation amount of CFU / mL The prodigiosin producing strain provided in Example 1 was inoculated into G2 medium, and fermented at 30° C. and 200 rpm for 48 h to obtain a fermentation culture solution.

[0073] The G2 culture medium specifically includes 10 g / L of glycerol, 3 g / L of peptone and 2 g / L of yeast powder.

[0074] S2. Purification of prodigiosin: (1) The fermentation culture solution was frozen at -20°C for 24 hours, then transferred to -80°C for a second freezing treatment for 24 hours, and finally vacuum dried at -80°C and 0.2 Pa for 48 hours to obtain a freeze-dried powder.

[0075] (2) The freeze-dried powder was mixed with acidic formaldehyde (pH = 2.5) at a ratio of 1 g:10 mL, and ultrasonication was performed for 10 min according to the procedure of ultrasonication for 2 s and rest for 8 s. The ultrasonication was repeated 3 times with an interval of 1 h between the two ultrasonications to obtain a cell-free extract; the cell-free extract was placed at 4 ° C in the dark for 24 h; and rotary evaporation was performed at a temperature of 40 ° C and an air pressure of 100 mbar for 30 min to obtain the substance to be extracted.

[0076] (3) The substance to be extracted was mixed evenly with distilled water in a ratio of 1 g:25 mL, and an equal volume of ethyl acetate was added for extraction. The extraction was repeated several times until the color of the aqueous phase became transparent, and the ethyl acetate phase was collected and subjected to rotary evaporation II for 60 min at a temperature of 45° C. and a pressure of 100 mbar to obtain an extracted dry matter.

[0077] (4) The extract dry matter was mixed with acidic formaldehyde (pH = 2.5) at an addition ratio of 1 g:10 mL, filtered using a 0.2 μm organic phase filter membrane, and the filtrate was subjected to rotary evaporation for 60 min at a temperature of 40° C. and an air pressure of 100 mbar to obtain prodigiosin.

[0078] S3. Determination of prodigiosin: (1) Dissolve 1 mg of prodigiosin in 1 mL of acidic methanol (pH = 2.5). Sampling and measuring the absorbance of the solution at 540 nm is 0.8. The concentration of prodigiosin in the solution is calculated based on the prodigiosin standard curve to be 1458.858 mg / L.

[0079] (2) Dissolve 1 mg of prodigiosin in 1 mL of acidic methanol (pH = 2.5) to obtain a test solution. Take chromatographic silica gel in a mortar and add the test solution dropwise. Grind each drop until the surface is fully filled so that the test solution is evenly dispersed in the chromatographic silica gel. Repeat several times until the test solution is completely added to obtain a volume of about 90 cm 3 Mixed silica gel; take the mixed silica gel and add it to the chromatographic tube pre-filled with silica gel for chromatography, add an appropriate amount of silica gel to seal the mouth, add 100mL of developing agent (n-hexane: ethyl acetate: formic acid = 2:1:0.01) for chromatographic column separation, and the obtained bands are as follows Figure 1 As shown; the eluents of different bands were taken for liquid chromatography analysis, and the results were as follows Figures 2 to 4 As shown. Figures 2 to 4 The purity of prodigiosin was calculated to be 9.08% and the mass was 138.45 mg based on the liquid chromatogram in FIG.

[0080] The conditions of liquid chromatography analysis include: elution program is 0-2min 10% methanol, 2-25min 50% methanol, 25-32min 100% methanol, 32-35min 50% methanol, 35-40min 10% methanol; the flow rate is 1mL / min.

[0081] (3) Take Figure 1 The eluate of band B was subjected to rotary evaporation at 40°C and 100 Pa for 30 min, and then vacuum dried at -80°C and 100 Pa for 24 h to obtain a lyophilized powder. 5 mg of the lyophilized powder was mixed with 1 mL of deuterated chloroform, filtered using a 0.2 μm organic phase filter membrane, and analyzed by mass spectrometry. The results are as follows: Figure 5 shown.

[0082] The conditions for mass spectrometry analysis include ion source mode: electrospray ionization (ESI) positive ion mode; scanning range: m / z 100-100; ionization voltage: +4.5 kV; gas flow rate: 10 L / min; and heating temperature: 200°C.

[0083] Depend on Figure 5 The test results show that the prodigiosin prepared in this example has the structure shown in formula (1).

[0084]

[0085] Example 3

[0086] This example is used to illustrate the chelating ability of prodigiosin provided in the above examples for iron ions. The test specifically includes:

[0087] 1. Chelating ability of prodigiosin for iron ions in solution

[0088] (1) Distilled water was mixed with the prodigiosin provided in Example 2 to prepare test solutions with concentrations of 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM and 1.0 μM, and each test solution was added to a 96-well plate at an addition amount of 100 μL / well, and a 2 mM FeCl2 solution and a 5 mM ferrozine solution were added to the 96-well plate at an addition amount of 2 μL / well and 4 μL / well, respectively, and the mixture was reacted at room temperature for 10 min;

[0089] (2) The absorbance of each well solution at 562 nm was measured by an ELISA instrument, and 100 μL of distilled water was used as a blank control. The ferrous ion chelation rate (%) was calculated according to the following formula. The test results are as follows: Figure 6 and 7 shown.

[0090] Ferrous ion chelation rate = [1-(sample absorbance / blank sample absorbance)] × 100%

[0091] Depend on Figure 6 and 7 The test results show that the prodigiosin provided in Example 2 has good chelating ability for ferrous ions, and the ferrous ion chelating rate increases with the increase of the solution mass concentration. When the prodigiosin concentration in the test solution is 1.0 μM, the ferrous ion chelating rate is 49.3%.

[0092] 2. Chelating ability of prodigiosin for intracellular iron ions in HEK293T cells

[0093] (1) According to 1.0×10 5 HEK293T cells were added to 10 mL of DMEM medium (Thermo Fisher Scientific, catalog number 11965092) at an inoculation volume of cells / mL, and incubated in a 37°C, 5% CO2 incubator for 24 h to obtain seed solution;

[0094] (2) According to 1.0×10 6 1 mL of seed solution was added to a 24-well plate, and RSL3 was added at a final concentration of 1 μM to treat HEK293T cells to induce iron overload;

[0095] (3) Distilled water was mixed with the prodigiosin provided in Example 2 to prepare test solutions with concentrations of 0.04 μM, 0.08 μM and 0.1 μM, and each test solution was added to a 24-well plate at an addition amount of 100 μL / well, and incubated in a 37° C., 5% CO2 incubator for 24 h, and 100 μL of PBS buffer (pH=7.4) was used as a blank control;

[0096] (4) Collect HEK293T cells from each well by centrifugation, add 0.2 mL of cell lysis buffer (ThermoFisher Scientific, catalog number 89900), mix well, place on ice for lysis for 10 min, centrifuge at 15,000 × g for 10 min, and collect the supernatant;

[0097] (5) 80 μL / well of the supernatant from each well was added to a 96-well plate and marked as a measurement well; 80 μL / well of a 0.5 mmol / L ferrous standard solution (Sigma-Aldrich, catalog number 372870-25G) was added to a 96-well plate and marked as a standard well; then 80 μL / well of a colorimetric solution (Sigma-Aldrich, catalog number 160601) was added to the measurement well and the standard well and mixed, and the mixture was incubated at 37°C for 40 min. The absorbance of the solution in each well at 593 nm was measured using an ELISA reader, and 100 μL of distilled water was used as a blank control. The relative ferrous ion content in the cell was calculated, and the ferrous ion chelation rate (%) was calculated according to the following formula. The test results are shown in FIG. Figure 8 shown.

[0098] Ferrous ion chelation rate = (1-A 标 ) / A 测 ×100%

[0099] Among them, A 标 is the absorbance value measured in the standard well, A 测 The absorbance value measured in the assay well.

[0100] Depend on Figure 8 The test results shown show that prodigiosin can alleviate the ferroptosis phenomenon caused by ferroptosis activators such as RSL3 by chelating with ferrous ions.

[0101] Example 4

[0102] This example is used to illustrate the inhibitory effect of prodigiosin on hepatocyte ferroptosis provided in the above examples. The test specifically includes:

[0103] 1. Effect of treatment time on the ferroptosis inhibition effect of prodigiosin

[0104] (1) According to 1.0×10 6 Normal human liver cells L02 were placed in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin, and cultured in a 37°C, 5% CO2 incubator. The medium was changed every 3 days. When the cells grew to 80% confluence, they were digested and passaged with trypsin to obtain seed solution.

[0105] (2) distilled water was mixed with the prodigiosin provided in Example 2 to prepare a test solution with a concentration of 1 μM;

[0106] (3) According to 5×10 3 The seed solution was added to a 96-well plate at an inoculation volume of 10 μL / well, and different concentrations of RSL3 were added to the 96-well plate at an inoculation volume of 10 μL / well, and cultured in a 37°C, 5% CO2 incubator for 24 h;

[0107] (4) The above test solution was added to a 96-well plate at a volume of 10 μL / well, and 3 replicate wells were set for each test solution. The plates were incubated in a 37°C, 5% CO2 incubator for 6 h and 24 h. Then, MTS (Zeye Biotechnology, catalog number ZY60102BB) was added to a 96-well plate at a volume of 10 μL / well, and the plates were incubated at room temperature for 3 h. The absorbance of the solution in each well at 490 nm was measured using an ELISA reader. 10 μL of DMEM culture medium was used to replace the test solution as a negative control group, and 2 μL of 100% (v / v) Triton X-100 was used to replace the test solution as a positive control. The cell survival rate (%) was calculated according to the following formula. The test results are shown in Table 1. Figures 10 to 15 shown.

[0108] Cell survival rate = [A x -A0] / [A1-A0]×100%

[0109] Among them, A x is the OD of the test solution group or the negative control group 490 A1 is the OD of the positive control group 490 A0 is the OD of the blank control group in which RSL3 and the test solution were replaced with an equal volume of DMEM medium in steps (3) and (4). 490 value.

[0110] Depend on Fig. 9 and 10 The test results shown show that, compared with the negative control group, the prodigiosin provided in Example 2 can effectively inhibit the ferroptosis of normal human liver cells L02 caused by RSL3, and the cell survival rate is significantly improved.

[0111] 2. Effect of treatment concentration on the ferroptosis inhibition effect of prodigiosin

[0112] (1) According to 1.0×10 6 Normal human liver cells L02 were placed in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin, and cultured in a 37°C, 5% CO2 incubator. The medium was changed every 3 days. When the cells grew to 80% confluence, they were digested and passaged with trypsin to obtain seed solution.

[0113] (2) mixing distilled water with the prodigiosin provided in Example 2 to prepare test solutions of different concentrations;

[0114] (3) According to 5×10 3 The seed solution was added to a 96-well plate at an inoculation volume of 100 μL / well, and RSL3 with a concentration of 1 μM was added to the 96-well plate at an inoculation volume of 100 μL / well, and cultured in a 37°C, 5% CO2 incubator for 24 h;

[0115] (4) The above test solution was added to a 96-well plate at a volume of 10 μL / well, and 3 replicate wells were set for each test solution. The plates were incubated in a 37°C, 5% CO2 incubator for 6 h. Then, MTS was added to a 96-well plate at a volume of 10 μL / well, and the plates were incubated at room temperature for 3 h. The absorbance of the solution in each well at 490 nm was measured using an ELISA reader. 10 μL of DMEM culture medium was used to replace RSL3 as a negative control group, and 2 μL of 100% (v / v) Triton X-100 was used to replace the test solution as a positive control. The cell survival rate (%) was calculated according to the following formula. The test results are shown in Table 1. Fig.11 shown.

[0116] Cell survival rate = [A x -A0] / [A1-A0]×100%

[0117] Among them, A x is the OD of the test solution group or the negative control group 490 A1 is the OD of the positive control group 490 A0 is the OD of the blank control group in which RSL3 and the test solution were replaced with an equal volume of DMEM medium in steps (3) and (4). 490 value.

[0118] Depend on Fig.11 The test results show that, compared with the negative control group, the prodigiosin provided in Example 2 can effectively inhibit the ferroptosis of normal human liver cells L02 caused by RSL3 within the concentration range of 0.1 to 10 μM, and the cell survival rate is significantly improved.

[0119] Example 5

[0120] This example is used to illustrate the effect of prodigiosin provided in the above examples on the expression of ferroptosis markers in hepatocytes. The test specifically includes:

[0121] (1) According to 1.0×10 6 Normal human liver cells L02 were placed in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin, and cultured in a 37°C, 5% CO2 incubator. The medium was changed every 3 days. When the cells grew to 80% confluence, they were digested and passaged with trypsin to obtain seed solution.

[0122] (2) According to 1.0×10 4 The seed solution was added to a 96-well plate at an inoculum volume of cells / well, and then 1 μM RSL3 was added to the 96-well plate at an addition volume of 10 μL / well, and cultured in a 37°C, 5% CO2 incubator for 24 h;

[0123] (3) distilled water was mixed with the prodigiosin provided in Example 2 to prepare test solutions with concentrations of 0.04 μM, 0.08 μM, and 0.1 μM;

[0124] (4) Add the above test solution to a 96-well plate at a volume of 10 μL / well, with 3 replicate wells for each test solution, and culture in a 37°C, 5% CO2 incubator for 24 h. In steps (3) and (4), replace RSL3 and the test solution with an equal volume of DMEM culture medium as a blank control group;

[0125] (5) Samples were taken from each well, and the iron ion content in the cells was detected using a cell iron ion detection kit (Elabscience Biotechnology Co., Ltd., catalog number E-BC-K139) according to the instructions. The relative increase in iron ions in each group was calculated based on the data measured in the blank control group. The test results are shown in Figure 2. Fig.12 As shown;

[0126] (6) Samples were taken from each well, and DCFH-DA (MedChemExpress, catalog number HY-D0940) was mixed with the sample at a final concentration of 5 μM, and incubated at 37°C for 20 min, with mixing once every 5 min. The cells were washed and collected with PBS, and the fluorescence brightness of the cells representing the ROS content was detected using a fluorescence microplate reader (Ex.492, Em.517). The relative fluorescence intensity of each group was calculated based on the data measured in the blank control group. The results are shown in Figure 2. Fig.13 As shown;

[0127] (7) Samples were taken from each well, and the MDA content in the cells was detected using an MDA kit (Biyuntian, catalog number S0131S) according to the instructions. The relative increase in MDA in each group was calculated based on the data measured in the blank control group. The test results are shown in Figure 2. Fig.14 As shown;

[0128] (8) Samples were taken from each well and the GSH content in the cells was detected using a GSH kit (Biyuntian, catalog number S0053) according to the instructions. The test results were as follows: Fig.15 shown.

[0129] Depend on Figures 12 to 15 The test results shown show that when prodigiosin acts on normal human liver cells L02, it can reduce the intracellular iron ion content by chelating with intracellular iron ions, and at the same time reduce the expression of three intracellular ferroptosis markers, namely ROS, MDA and GSH, thereby achieving an inhibitory effect on cell ferroptosis.

[0130] Example 6

[0131] This example is used to illustrate the alleviating effect of prodigiosin provided in the above examples on non-alcoholic fatty liver disease. The test specifically includes:

[0132] (1) Fifty male C57BL / 6J mice were used as experimental animals and randomly divided into a normal control group, a nonalcoholic fatty liver disease model group, and an experimental group (low-dose, medium-dose, and high-dose groups), with 10 mice in each group. The mice were fed for 4 weeks according to Table 2.

[0133] Table 2.

[0134]

[0135] (2) After the feeding period, fasting blood samples were collected from each group of mice, and the blood samples were tested for liver function-related indicators. The results were as follows: Figures 16 to 24 shown.

[0136] Depend on Figures 16 to 24 The test results shown show that compared with the mice in the non-alcoholic fatty liver model group, after the administration of 3mg / (kgd) of prodigiosin to mice, multiple indicators such as ALT concentration, AST concentration, LDH concentration and HA concentration in the blood samples of mice were restored, which has a good effect in alleviating non-alcoholic fatty liver inflammation. Among them, prodigiosin can effectively improve the liver damage induced by choline-deficient diet in mice, effectively reduce ALT and HA levels, and restore PIICP expression, which has good liver protection and anti-fibrosis effects.

[0137] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention.

Claims

1. A prodigiosin producing strain, characterized in that: The production strain is Serratia marcescens MMOG2448, and the deposit number of the Serratia marcescens MMOG2448 is GDMCC 65757.

2. A method for preparing prodigiosin, characterized in that: The preparation method comprises: S1, taking the prodigiosin production strain of claim 1 for fermentation culture to obtain a fermentation culture liquid; S2, taking the fermentation culture liquid for purification treatment to obtain the prodigiosin.

3. The preparation method of prodigiosin according to claim 2, characterized in that, In step S1, the medium for fermentation culture is selected from one or more of G2 medium, PG medium, NB medium, NBM medium, PBO medium, SP medium, GPK medium, and Brown medium; Optionally, the inoculation amount of the prodigiosin producing strain is 10-10 CFU / mL; Optionally, the fermentation culture temperature is 25 to 30° C. and the fermentation time is 24 to 72 hours.

4. The preparation method of prodigiosin according to claim 2, characterized in that, In step S2, the purification treatment includes: (1) taking the fermentation culture liquid for freeze-drying treatment to obtain freeze-dried powder; (2) taking the freeze-dried powder and mixing it with acidic formaldehyde, performing ultrasonic crushing, light-proofing and standing treatment and rotary evaporation treatment I to obtain the substance to be extracted; (3) taking the substance to be extracted and mixing it with water to obtain the liquid to be extracted, taking the liquid to be extracted and the extractant for extraction treatment and rotary evaporation treatment II to obtain the extracted dry matter; (4) taking the extracted dry matter and mixing it with acidic formaldehyde, filtering treatment and rotary evaporation treatment III to obtain the prodigiosin.

5. The method for preparing prodigiosin according to claim 4, characterized in that, In step (1), the freeze-drying treatment includes: taking the fermentation culture liquid for a first freezing treatment at -25 to -18°C for 12 to 48 hours, performing a second freezing treatment at -85 to -75°C for 12 to 48 hours, and then vacuum drying at -85 to -75°C and 0.1 to 0.2 Pa for 48 to 72 hours to obtain the freeze-dried powder.

6. The method for preparing prodigiosin according to claim 4, characterized in that In step (2), the pH value of the acidic formaldehyde is 2 to 3.5; Optionally, the added mass volume ratio of the lyophilized powder to the acidic formaldehyde is 1 g: (5-20) mL; Optionally, the temperature of the dark-proof static treatment is 0 to 5° C., and the time is 12 to 48 hours; Optionally, the temperature of the rotary evaporation treatment I is 30-40° C., the air pressure is 100-200 mbar, and the time is 30-60 min.

7. The method for preparing prodigiosin according to claim 4, characterized in that, In step (3), the added mass volume ratio of the substance to be extracted and water is 1 g: (10-50) mL, and the added volume ratio of the liquid to be extracted and the extractant is 1: (0.8-1.2); Optionally, the temperature of the rotary evaporation treatment II is 30-40° C., the air pressure is 100-200 mbar, and the time is 60-120 min; Optionally, in step (4), the pH value of the acidic formaldehyde is 2.0 to 3.5; Optionally, the added mass volume ratio of the extracted dry matter to the acidic formaldehyde is 1 g: (5-20) mL; Optionally, the temperature of the rotary evaporation treatment III is 30-40° C., the air pressure is 100-200 mbar, and the time is 60-90 min.

8. A prodigiosin, characterized in that The prodigiosin is prepared by the preparation method of prodigiosin according to any one of claims 2 to 7.

9. Use of the prodigiosin according to claim 8 in ferroptosis inhibitors.

10. Use of the prodigiosin according to claim 8 in liver protection drugs and / or health products.

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